Cell signaling is part of a vital communications network allowing cells to perceive and correctly respond to their microenvironment it is the basis of development, tissue repair, and cell functioning. Signals are generated, received, and transcribed by intracellular, transcellular and extracellular enzymes and proteins. It has been recently shown that the majority of proteins and enzymes in a mammal contain functional thiol (sulfur) groups, mainly in the form of the amino acid cysteine. These proteins and enzymes communicate with each other through redox reactions. In a redox reaction there are always two simultaneous reactions; an oxidation and a reduction. These redox sensitive thiol reactions are involved in almost every aspect of life, such as; energy regulation, cytoskeletal structure, transport, proliferation, differentiation and apoptosis. Without this communication life could not continue.
New dogma states that oxidative stress and chronic inflammation is the results of the body's inability to prevent the disruption of thiol circuits (Jones 2008). When non-radical compounds like hydrogen peroxide interfere with this communication flow, that is, they steal electrons, unbalancing cellular homeostasis. Once unbalanced, cells begin to produce pro-oxidant compounds including more hydrogen peroxide, resulting in additional oxidative stress. One of the major transcriptional pathways that produced pro-oxidants is the Nuclear Factor Kappa-Beta (NF-κB) pathway. This entire cascade of events produces a negative feedback loop within a cell or specific cells that can be local or systemic in an organ or the entire body. This negative feedback results in low level chronic inflammation, which then progresses over time into some form of degenerative or so called age related disease, such as atherosclerosis, cardiovascular diseases (CVDs), cancer, Alzheimer's disease, Type 1 and 2 Diabetes, liver cirrhosis or liver disease, Rheumatoid arthritis, osteoporosis, Irritable Bowl Syndrome, cataract disease, cystic fibrosis, asthma, hypertension, dementia, Parkinson's disease, gout, multiple sclerosis (MS), Lupis, and sepsis.
In this chronic inflammation state, the stated thiol(s) are either losing electrons to non-radical compounds or they are in a dormant non-reactive state because they have been protected by s-glutathiolation. Glutathiolation has emerged as an important post-translational modification that prevents irreversible oxidation of protein thiols, and recent evidence suggests that controlled glutathiolation reactions can also be used to modify protein structure and function. Reduced glutathione remains bound to reactive cysteine side chains of several intracellular proteins even under basal conditions and the abundance of glutathiolated proteins increases upon oxidant challenge (Hill & Bhatnagar, Role of glutathiolation in preservation, restoration and regulation of protein function. IUBMB Life. 2007 January; 59(1):21-6). These glutathiolated thiols can also be in a disulfide state, where bridging with other enzyme and proteins produces a disulfide bond. Also while in this state both proteins and enzymes can be incorrectly folded (via internal disulfide bonds). Regardless, in these states they are non-functional or they are functioning in a negative was towards life.
Recently, Resveratrol has been shown to be inhibited by S-nitrosoglutathione (GSNO) which resulted in S-gutathiolation of sirtuin-1 (Zee R S, Antioxidant Redox Signaling: 2010 online prior to publication). Since the small molecule complexes of this invention regulate thiol redox and thiol redox drives S-glutathiolation a conclusion can be drawn that this invention will help promote the therapeutic activities of Resveratrol by preventing and/or reducing S-glutathiolation. Following this logic the complexes in this invention can be used to enhance drugs and supplements that are subject to S-glutathiolation either directly or within the cellular pathway they are targeted for.
In the regulation of thiol redox it should be noted that this also extends to cover proteins and enzymes that are inactivated through S-glutathiolation/S-glutathionylation. A list of some of these proteins include actin, spectrin, tubulin, vimentin, glyceraldehyde-3phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, triose phosephate insomerase, pyruvate kinase, aldolase, alpha-ketoglutarate dehydrogenase, mitochondiral isocritrate, dehydrogenase, complex 1, NADHP, ATPase, NADH ubiquinone reductase, carbonic anhydrase III, catechol-O-methyltransferase, pyruvate dehydrogenase, MEKK1 (JNK), protein tyrosine phosphatase 1B, PTEN, pyrophosphatase 2A, Nuclear Factor Kappa Beta (NF-kB) subunits 65 and 50, PKC, PKG, (cAMP) dependent PKA, creatinine kinase, c-able, p53, caspase 3, GTPase p21 ras, S1000A1 and S100B, SERCA, ryanodine receptor I and II, CTFR, PDI, HSP 65,70, 20S proteosome, ubiquitin conjugating enzyme, thioredoxin 1, glutathione S-transferase An example some of the diseases associate with S-glutathiolation are listed in Table 1.
TABLE 1A correlation of diseases related to different protein dysfunction in humans.ProteinDiseaseActinCardiovascular/IschmiaTauAlzheimer's diseaseHemoglobinType 2 diabetesCTFRCystic Fibrosisγ-S-crystallinCataract diseaseSpectrinSickle cell anemia
It has been discovered that Glutathione (GSH) is responsible for maintaining thiol redox in mammals. GSH is found throughout the body and with age or exposure to xenobiotic (foreign) compounds (organisms, such as bacteria, viruses, or carcinogens, or other compounds the body does not make) decreases. Decreases in intracellular GSH have been shown to be directly related to disease states and or progression to disease states such as; Cardiovascular Diseases, Autoimmune Diseases, Pulmonary Diseases, Cystic Fibrosis, Lupus, Crohn's Disease, Type I Diabetes, Type II Diabetes/Diabetes Mellitus, Psoriasis, Contact Dermatitis, Multiple Sclerosis, Liver Diseases, and Viral Infections. (Sedda 2008) and (Ballatori 2009) Therefore, increasing intracellular levels of GSH through therapeutic means is desirable. GSH is produced by the intracellular enzyme Gamma-Glutamyl-cysteine synthase (GCS). GCS, however, can only be produced through DNA transcription, via the Nuclear Factor-Erythroid 2 p45-related Factor (Nrf2) pathway. Importantly, GCS is the rate limiting enzyme in GSH production. However, basic activation of Nrf2 doesn't guarantee the transcription of GCS.
While GCS is the rate limiting enzyme for the production of GSH the enzyme Gamma Glutamyltransferase, also know as Gamma Glutamyl Transpeptidase (GGT), is also involved in GSH production. GGT is a membrane bound enzyme that plays a key role in GSH production by transporting the required building block amino acids into the cell. It also transports GSH conjugated xenbiotic compounds out the cell. Importantly, the increase expression of GGT has been shown to be directly associated with a decrease in GSH while a decrease in GGT is associated with GSH increases or a steady state. (Zhang 2005, Ballatori 2009) GGT is therefore, directly involved in the regulation of thiol redox via the glutathione cycle. Thiol redox regulation is required to ensure that cell signaling pathways are maintained. Dysfunction of cell signaling leads to the pathogenesis of many human diseases (Yardimci, Clincal and Applied Thrombosis, 1995:1:2:103-113).
GGT has been used as a biomarker for predicting alcohol abuse and bile tract obstruction for over 30 years. However, modern research into oxidative stress and cell signaling has discovered that this enzyme has been misclassified, overlook and underutilized. In fact, GGT has become a primary biomarker for oxidative stress and for all cause of mortality. Elevations in GGT are seen in almost all chronic and life-style related diseases, such as oxidative stress, metabolic syndrome, Type II diabetes, fatty liver disease, cardiovascular diseases, Alzheimer's disease, liver cirrhosis, chronic kidney disease and cancer (Mason, Preventive Cardiology 2010: 13:1:36-41; Stojakovic, Atherosclerosis 2010: 208:2:564-571; Breitling, Atherosclerosis 2010: Jan. 11 Online 2010; Shimizu, Stroke AHA 2010: 41:385-388; Franzini, Medicine 2010: Online February 2010; Sen, Turkish Society of Cardiology 2009: 37:168-173; Poelzl, Circulation: Heart Failure 2009: 2:294-302; Korantzopoulos, Archives of Medical Research 2009: 40:7:582-589; Turgut, Archives of Medical Research 2009: 40:4:381-320; Emdin, International Journal of Cardiology, 2009: 136:1:80-85; Lee, International Journal of Clincal Chemistry 2009; 408:1-2; Abigail, Diabetes Care 2009: 32:4741-750.). Recently, the National Institutes of Health (NIH) has shown that elevated levels of GGT increase all cause mortality death rates in the United States (US) (Constance, Gasteroenterology 2009: 136:477-485). In fact, one of the most negative trends associated with elevated levels of GGT is its association with death. More importantly, GGT has been discovered to play a key role in the pathogenesis and progression of Atherosclerosis and Osteoporosis.
Recently, it has been discovered that GGT acts as a cytokine (a signaling molecule) when it comes to the initiation of osteoclast production. Mice genetically modified to over express GGT have major bone deformities, are dwarfs and died early as a result of accelerated osteoporosis Importantly, when collagen induced arthritic mice were treated with an Anti-Glutamyl Transpeptidase Antibody, osteoporosis was attenuated and in some cases reversed. In humans, extreme elevated levels of GGT are found in individuals with primary biliary cirrhosis (PBC). These individuals have an increased risk of Osteoporosis. Alcoholics can also have elevated levels of GGT and the prevalence of osteoporosis is higher in alcoholics. Interestingly, bone loss is halted or is reversed in alcoholics that abstain for up to 6 months.
Oxidized LDL (ox-LDL) is known to initiate the pathogenesis of Atherosclerosis. In blood serum, GGT is found unbound or it can be found bound to lipoproteins. Importantly, it has been shown that GGT oxidizes LDL from its enzymatic activity: Cisteinly-Glycine+Iron forms hydrogen peroxide which oxidizes LDL and activates NF-kB, a pro-oxidant intracellular pathway. By oxidizing LDL GGT becomes a direct participant in the pathogenesis of atherosclerosis. Additionally, increased oxidized LDL levels also increase Lipoprotein (a) (Lp(a)) levels. Lp(a) is a known confounding biomarker for Cardiovascular Diseases.
Finally, GGT has been discovered in arterial plaque. Once embedded in plaque it not only continues to oxidize LDL, but it is capable of signaling osteoclast development which leads to calcification. Increases in ox-LDL increases Lp(a) levels and individuals with elevated levels of Lp(a) have an increased risk of coronary artery calcification. Interestingly, elevated levels of GGT in individuals with recent coronary stent implantation indicate stent failure. Also, serum GGT is predictive of all-cause and cardiovascular death in individuals with coronary artery disease (CAD) independently of other cardiovascular risk factors.
Combining the above three GGT discoveries sets up a chain of events that are simultaneously detrimental to both bone and vascular health. As noted above, GGT oxidizes LDL, which increases Lp(a) and initiates osteoporosis. GGT also increases osteoclast development, accelerating bone loss. As atherosclerosis progresses, plaques are formed, which are attacked by the increased osteoclast population, resulting in the calcification of the plaque and increase in mortality. The interaction is cyclical, further propogating the disease.
Taking the above discussion into account, a method to reduce GGT would be a major step in assisting with slowing the initiation and progression of Atherosclerosis, Osteoporosis and possible other vascular and inflammatory diseases such as Type II Diabetes. At this time, there are no FDA approved drugs or supplements that can significantly reduce GGT. In fact, as xenobiotic compounds the basic metabolism of drugs and supplements actually produces oxidative stress and thus decreases the intracellular levels of GSH which increases GGT expression. Since GGT levels are directly associated with GSH levels and GSH levels are tied to the rate limiting transcriptional enzyme GCS the focus on reducing GGT expression should be centered on increasing the expression of GCS.
Most xenobiotic compounds will temporarily increase GSH, via a cascade of events that activates/simulates/signals GCS to produce GSH. These xenobiotic compounds are seen as invaders to the cell and therefore GSH production is increased. This increase however, can't be sustained unless the rate-limiting enzyme GCS is increased (transcribed/expressed through the Nrf2 pathway) prior to GSH production. If GCS is not increased prior to GSH the cell is placed in a lag phase where the production of GSH is always lagging behind the concentration of whatever compound(s) are attacking the cell. This lag phase produces an environment where non-radicals continue to disrupt thiol signaling. In this lag phase GSH level drop and upstream expression of GGT increases. Most importantly, in this state cross talk between multiple transcriptional pathway is lost or misdirected leaving some pathways to constantly transcribe compounds. For example in this state the NF-κB pathway may continually produce pro-oxidants that in turn continually add to the non-radical load of the cell. On the other hand, the Nrf2 pathway can be silent, thereby preventing production of oxidation-protective enzymes. Ultimately the homeostasis of the cell's thiol signaling is lost.
To realistically increase GSH, the rate-limiting enzyme GCS must be increased (transcribed) prior to any increases in GSH. Since xenobiotic compounds increase GSH it is difficult to find therapeutic compounds that increase GCS prior to GSH production. Hundreds of compounds have been tested around this requirement. Of all the compounds tested, in the available literature, only two compounds have been identified that increase GSC prior to GSH. Those two compounds are the small therapeutic molecules (STMs) Cafestol and Kahweol (C&K) (Scharf 2003).
C&K are diterpenes which may be isolated from the oil of the coffee bean, where they are found bonded to a single fatty acid group (they are esterfied). These fatty acids can vary by type, such as C14, C16, C18, C18:1, C18:2, C18:3, C20, C22 and C24. In their pure forms they are unstable and susceptible to breakdown from heat. Interestingly, just as GGT has been discovered to be associated with the increased risk in Cardiovascular Disease, Fatty Liver Disease, Type II Diabetes, Metabolic Syndrome, Liver Cirrhosis, Alzheimer's/Dementia and Cancer, the consumption of coffee at 6 or more cups per day has been shown to reduce the risk of these same diseases. Studies indicate that drinking 6 or more cups per day increase overall life spans or decrease overall morality rates, and high coffee consumption has also been shown to reduce both GGT and Lp(a) by up to 15%. C & K were identified in coffee in the late 1980s. During this period, methods of coffee preparation were also tested to identify the amounts of C & K contained in each extraction method, seen in Table 2.
TABLE 2The concentration of extracted from coffee beans using various extraction methods. The tests were based on 180 ml water and 10 grains of ground coffee beans.Method of Extraction Amount of C & K (mg)Filtered0.2Percolated0.2Instant0.4Espresso3.3Mocha2.5Boiled6.9French7.9Turkish/Greek7.8
As seen in Table 2 filtered coffee was shown to have very little C & K in it; 0.2 mg per cup, mainly because the filter removes any of the oil extracted from the coffee bean. This may be the primary reason it take up to 6 cups of coffee per day to see the health benefits of drinking coffee. It should be noted that hot water or steam alone is not an effective method for the removal/extraction of the hydrophobic compounds found in the coffee bean. Though not seen in Table 2, when pure coffee oil is extracted from 10 grams of ground coffee beans using an organic solvent, such as hexane, the analysis of the coffee oil indicates that it contains approximately 1800 mg of C & K, or about 9000 times the amount found in one cup of filtered coffee.
Cafestol and Kahweol have been identified by several laboratory research studies to upregulate Nrft2, which conjugates toxic compounds and removes them from the cell; and to downregulate NF-κB, which prevents the expression of chronic inflammation compounds such as COX-2 or iNOS. The disclosed compositions will logically possess the same characteristics, such as limiting diseases that are connected to each of these pathways, such as, cancer caused by exposure to toxic environmental compounds.
C&K activate or upregulate Nrf2 which signals the DNA to produces Phase II enzymes, such as GCS, responsible for generating GSH; Glutathione S-Tranferase (GST), which generates GST-P. GST-As, GST-Ms; Quinon Oxidoreductases such as NQO1 or NADPH; UDP-Glucoronosyl-Transferase (UGT); Heme Oxygenase-1 (HO1); sulfotransferases [SULTs]; acetyltransferases [NAT1&2]; and methyl transferase. These Phase II enzymes are known to be cancer preventative proteins involved in the detoxification (conjugation) of carcinogenic xenobiotic compounds, some of which are persistent organic pollutants (POPs) that are encountered through diet or other exposure methods such as breathing and/or adsorption.
Laboratory studies have shown that C&K are protective against carcinogens or suspected carcinogens such as carbon tetrachloride; azoxymethane; N-Nitrosodimethylamine (NDMA), an industrial byproduct from rocket fuel found as a low-level contaminant in food and in water; acrolein, which is a byproduct from degradation of tobacco, gasoline, fried food, and cooking oil; HeteroCyclic Aromatic Amines (HAA or HCA), which are a degradation byproduct of foods prepared with heat; pyridine (PhIP), found in cooked meat and fish; benzo[α]pyrene, found in coal tar, tobacco smoke, charbroiled foods and burnt toast; aflatoxin B1, a mold toxin found in spoiled grains; 6-OHDA, a causative Parkinson's disease agent in laboratory mice. It is noted that any compound that can reduce the damage from CCL4 may have a significant role in maintaining health when used as medicine or consumed as a part of the normal diet.
C&K also inhibit or down-regulate the transcription factor NF-κB, which is responsible for signaling the DNA to produce pro-oxidant enzymes such as inducible Nitric Oxide Synthase (iNOS), Prostaglandin E Synthase-2 (PGE-2) and Cyclooxygenase-2 (COX-2). These destructive enzymes are part of a response that keep cells proliferating and protects them from apoptosis. However, acute inflammation process can become chronic with constant activation of NF-κB. In fact elevated levels of iNOS, PGE-2, COX-2 and NF-κB are associated with Chronic Inflammatory Diseases, such as; Alzheimer's disease, Parkinson's disease, atherosclerosis, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, sepsis, osteoporosis autoimmune disease, asthma, hypersensitivities (such as allergies), pelvic inflammatory disease, Rosacea, transplant rejection, and other chronic inflammatory diseases. Additionally, Chronic Inflammation has recently been shown to be directly linked to cancer.
C&K are also intracellular and extracellular SuperOxide Scavengers, that is they are involved in reducing non-radicals such as hydrogen peroxide and tert-Butyl hyperoxide.
Ironically, though C&K can increase GCS prior to GSH demand, act as intracellular antioxidants, upregulate Nrf2 Phase II enzyme production and downregulate, NF-κB pro-oxidant production, these compounds have been shown to also increase cholesterol and damage the liver. Testing of C&K in individuals who consumed unfiltered coffee, coffee oil or coffee grinds revealed that C&K increased cholesterol and liver damage. Increases in cholesterol have been associated with increased in cardiovascular diseases (CVDs), while liver damage leads to possible cirrhosis, liver failure, and death. Recently, in the mouse model Cafestol was shown to be an agonist ligand for Farnesoid X Receptors (FXR). (Ricketts 2007) These receptors are located in the distal portion or ileum portion of the small intestine in most mammals including humans. Activation of FXR activates the recirculation of bile acids which can increase cholesterol and damage the liver.
In a 1996 study, coffee and coffee oil containing high concentrations of C&K raised the liver inflammation enzyme alanine aminotransferase (ALT) by 35 units per liter (U/L) on average in test subjects. In fact several subjects were removed from the study because their elevations were above the ALT cutoff level 53.5 U/L. Low density lipoprotein cholesterol concentrations in subjects rose by 9-14% relative to filtered coffee and triglyceride concentrations initially rose by 26%, but returned close to baseline within 6 months (Urgert, Br Med J, 1996). Coffee oil (Arabica), in a 2003 study on liver enzymes, showed that C&K dramatically raised ALT and aspartate aminotraseferase (AST) levels. In this study extreme ALT elevation were observed at 3.6, 5.8 & 12.4 times the cutoff of 45U/L, while AST elevations were observed at 2.0 and 4.7 times the cutoff of 50 U/L. (Boekschoten 2004). These facts prompted the medical community and certain government agencies throughout the world to warn people about the dangers of drinking unfiltered coffee and consuming coffee oil. Ultimately, the use of C&K or coffee oil as supplements to regulate thiol redox via the amplification of GCS along with the amplification of the Nrf2 pathway has been ignored because of the stated negative biological issues in humans. As discussed earlier C&K amplify the Nrf2 pathway thus they increase the transcription of many different Phase II enzymes such as the Glutathione Transferases Family which are involved in detoxification by conjugating xenobiotic/toxic electrophiles to GSH for their removal from the cell. Because these Phase II enzymes required GSH amplifying their expressions does not provide health benefits to the cell if GSH is not increased through the amplification of the expression of GCS. Without this type of increase the Phase II enzymes will use up the intracellular GSH pool. Therefore, targeting GCS for amplification is also required for Phase II enzyme optimization.
However, there are currently no acceptable compounds for modulating GCS prior to GSH demand and preventing and correcting diseases associated with GCS reduction, GSH reduction and GGT increases. Accordingly, the present invention addresses these important needs.